Acidic phallotoxin derivatives and methods of preparation

ABSTRACT

The present invention relates to novel compounds which are derivatives of acidic phallotoxins and correspond to the formula: ##STR1## (NBD-acid phallotoxins) their preparation, and their use in fluorescent staining of F-actin.

The U.S. Government has right in this invention pursuant to Grant Nos.GM 21661-05 and CA 14454 and PCM 75-83068 awarded by the Department ofHealth, Education and Welfare and th National Science Foundation,respectively.

This is a division of application Ser. No. 124,208, filed Feb. 25, 1980now U.S. Pat. No. 4,387,088.

The present invention relates to NBD-acidic phallotoxins, activefluorescent derivatives of the actin-binding mushroom toxins, such ofPhallacidin, Phallisacin, Phallacin, etc., and to their synthesis. Thepresent invention also relates to methods for staining F-actin, forexample in cytoskeletal structures in living and fixed cultured animalcells and actively streaming algal cells. Actin binding specifically wasdemonstrated by competitive binding experiments and comparative stainingof well known structures. Large populations of living animal cells inculture were readily stained using a relatively mild lysolecithinpermeabilization procedure facilitated by the small molecular size ofthe label. F-actin in animal cells was stained in stress fibers,ruffles, the cellular geodome and in diffuse appearing distributionsapparently associated with the plasma membrane. Staining of F-actincables in algae with the NBD-acidic phallotoxins of the invention didnot inhibit cytoplasmic streaming. NBD-acidic phallotoxins provide aconvenient actin-specific fluorescent label for cellular cytoskeletalstructures with promise for use in studies of actin dynamics in livingsystems.

Topographical fluorescence microscopy images of the major features ofthe cellular cytoskeleton have advanced the knowledge of cytoskeletalstructures during the last few years. Labeling of fixed cells byindirect immunofluorescence with anti-actin antibodies or by fluorescentheavy meromyosin has been used to study microfilaments and theirrelationship with other cytoskeletal components. More recently,microinjection of fluorescent actin has been used to study thesestructures in living cells.

The present invention relates to a novel fluorescence stainingcomposition which is used as a fluorescent marker for cellular F-actinand G-actin oligomers. It is applicable to living cells and therebyoffers potential for observing the dynamics of cellular processes.

The acid phallotoxins derivatives of the present invention can beemployed as fluorescent markers for actin which can be introducedconveniently into large populations of living cells, unlike fluorescentantibodies and myosin derivatives. Phallotoxins, small cyclic peptideshave been shown previously to bind specifically and to stabilizeF-actin, see T. Wieland and H. Faulstitch, CRC Critical Reviews inBiochemistry, 5, 184 (1978). In particular the acidic phallotoxin,phallacidin, that abounds in American strains of the poisonous mushroomAmanita phalloides offers small molecular size (molecular weight of 847)and a convenient carboxylic acid residue for attachment of thefluorescent label as described by R. R. Yocum and D. M. Simons inLloydia, 40 (2), 178-179 (1977). Although this toxin differs in thiscarboxylic acid residue from phalloidin, its actin bindingcharacteristics are essentially identical.

SYNTHESIS OF AN NBD DERIVATIVE OF PHALLACIDIN

Phallacidin was purified by column chromatography from a mixture ofmushroom toxins residual to an amanitin purification. This purificationis well known to one skilled in the art as exemplified in a procedure byR. R. Yocum in Biochemistry, 17 (18), 3786-9 (1978). Phallacidin isrepresented by structural formula I. ##STR2## wherein R is --OH, and R₂is H and R₁ and R₃ are --OH.

The phallacidin was obtained as a byproduct of an amanitin purification.After the first neutroal LH-20 Sephadex (Pharmacia) chromatography, thephallacidin fraction was still contaminated with a dark brown polarmaterial. Three additional steps A, B and C removed most contaminantsproviding purified phallacidin toxin.

In step A, crude phallacidin plus phallisacin from 100 gm of A.Phalloides were chromatographed on a 25 mm×200 cm LH-20 column in 0.1 Nacetic acid. Phallacidin eluted between fractions 125-150. 1.5 ml ofthis crude fraction containing approximately 60 mg of toxin was placedon a 1.5 cm×400 mm LH-20 column and eluted with 0.08N acetic acid at 7ml/hr. The majority of brown material eluted in the early fractions,whereas the phallacidin eluted between 78 and 110 ml. The pooledphallacidin fractions were neutralized with ammonium hydroxide to pH7.0, reduced to a volume of 1 ml on a rotovaporizer, desalted on aSephadex G-25 column and chromatographed a second time on LH-20.

In step B, the toxin fraction obtained from step A was applied to QAESephadex prepared as described by Yocum in a 1.1 cm×15 cm column. Astepwise salt gradient of 0.05N NaCl intervals was used to develop thecolumn. The phallacidin eluted between 0.05 and 0.3 N NaCl leaving brownmaterial remaining on the column. The toxin was again concentrated anddesalted as in step A above.

SP-50 Sephadex was equilibrated with 0.5N ammonium acetate at pH 6.5 andthen washed with 5 volumes of water in a Buchner funnel. The Sephadexwas then placed in a column and washed with two more volumes of water.The concentrated material from B was placed on a 1.5 cm×5 cm cationexchange column of SP-50 Sephadex. The column was first eluted withwater. Phallacidin, which eluted in the flow through, was then appliedto Amberlite XAD-4 (Rohm & Haas) in a 2 cm×5 cm Buchner funnel fittedwith a 25-50 μm fritted-glass disk. The amberlite had been cleaned withsequential 8-12 hour washes in 1N NaOH, 1N HCl, chloroform, 95% ethanol,and water. The amberlite was washed with 40 ml of distilled water andthen the toxin was eluted with 40 ml of absolute ethanol. The toxinsolution had a slight brown color after being concentrated to 1 ml.However, no impurities were detectable as alterations in the phallacidin240-320 nm absorption spectrum

The following steps produce the desired phallicidin derivative from thepurified phallicidin obtained in step C.

In step D, the toxin solution from step C was dried in a rotovaporizerand redissolved in dimethylforamide (DMF) at a concentration of 56mg/ml. The solution was placed over dry ice and an excess ofdiazomethane in either was added at 76° C. in a well ventilated hood.The mixture was allowed to warm up to room temperature for 5-10 minutesafter which the DMF and diazomethane were removed under high vacuum in arotovaporizer. This converted the phallacidin carboxylic acid residue toa methyl ester as represented in Formula I wherein R is --OCH₃.

The dried material was redissolved in water and a fraction was removedfor thin layer chromatography (TLC). TLC showed that 50-60% of the toxinhad been altered. The toxin solution was redried, dissolved inethylenediamine and left at room temperature for 90 minutes. The excessethylenediamine was removed under reduced pressure and the driedmaterial was placed in 1 ml of water at 4° C. for 1.5 hours.

The toxin solution was placed on an SP-50 Sephadex column equilibratedwith 0.5N ammonium acetate at pH 6.5. The material at this timecontained a yellow-green impurity Upon application of the toxin solutionto the SP-50 column, a colored fraction eluted in the flow-throughwhereas another remained bound to the column and eluted between 0.1-0.5NNaCl. This second fraction contained the ethylenediamine-linked materialas represented by Formula I wherein R is --NH--CH₂ --CH₂ --NH₂. Theyield of the reaction as shown by UV absorption at 292 nm was 54% usingε292=1.1×10⁴ /1-M-cm.

The ethylenediamine phallacidin (N-Ph) fraction was desalted onamberlite, concentrated, and placed on a 1.1 cm×24 cm LH-20 Sephadexcolumn equilibrated with 0.02N acetic acid. The column was developed at3.5 ml/hr. Greenish toxin containing material eluted earlier than thepure ethylenediamine-linked derivative found between 14 and 27 ml. Thepurified toxin fractions were pooled, desalted on amberlite, redissolvedin water and lyophilized. There were stored dessicated at 4° C. untillater use. The final purification yielded 7.7 mg of pure material asshown by UV absorption.

In step E, approximately 200 μg of the purified material of step D wasdissolved in 200 μl of absolute methanol plus 4 μl of anhydrouspyridine. 50 μl of freshly prepared 10 mg/ml4-chloro-7-nitrobenz-5-oxa-1,3-diazole, (NBD-Cl) (Molecular Probes) inmethanol was added. The materials reacted for 12 minutes at 70° C. overan oil bath. The solution was reduced to 50 μl with dry nitrogen andallowed to react for 5 minutes more at 50° C. 600 μl of diethylether wasadded to the solution and a brownish precipitate formed. The solutionwas centrifuged and the supernatant removed. The toxin containingprecipitate was resuspended in 20-30 μl of absolute methanol and theprocedure repeated three more times with 400-600 μl of diethylether perextraction to remove free NBD-Cl.

In step F, Sp-50 Sephadex was prepared as in step C. TheNBD-ethylenediamine phallacidin (hereinafter NBD-phallacidin or NBD-Ph)was dissolved in 200 μl of water and placed on the column. Yellowcolored material, the NBD-Ph, eluted in the flow-through whereasunreacted toxin and brownish material, probably a salt of pyridine andNBD-Cl, eluted in the NaCl fractions. The NBD-phallacidin (NBD-Ph) isrepresented by structural Formula II: ##STR3## wherein R₂ is H, and R₁and R₃ are --OH.

Step G represents equilibrium dialysis experiments which were performedin order to determine whether the N-Ph bound to actin. Buffer Aconsisting of 5 MM Tris HCl (pH 7.4) and 0.1 mM ATP and buffer Bconsisting of buffer A plus 2 mM MgCl₂ were prepared immediately beforeuse. 3.05 mg actin (Sigma) and an equal amount of 5X crystalizedovalbumin (Calbiochem) were dissolved in separate volumes of 2 ml ofbuffer A. The fractions were split into four 1 ml aliquots 21 μg of N-Phin 1 ml of buffer was added to each of the four opposing compartments inthe dialysis chambers. Dialysis was performed overnight at 4° C.

In step H, in order to assess whether the amino-linked phallacidinenhanced actin polymerization, the viscosity of solutions containingN-Ph was measured. Viscosity measurements were performed in an OstwaldDropping Pipette with a 2 ml volume. The viscometer was cleaned beforeeach measurement. The dialyzed actin and toxin containing chambers as instep G were diluted to 1/3 their concentration with buffer B. Relativeviscosities were determined from the average of 3-5 measurements.

                  TABLE II                                                        ______________________________________                                        Viscosity Measurements of Actin                                               Solutions                                                                     Solution       Relative Viscosity                                             ______________________________________                                        1.       Water     1                                                          2.       `1` + Tris                                                                              1                                                          3.       `2` + Toxin                                                                             1                                                          4.       Actin + `2`                                                                             1.11                                                       5.       `4` + Toxin                                                                             1.25                                                       ______________________________________                                    

In step I, Thin Layer Chromatography (TLC) was performed on the variousphallacidin derivatives. All samples were run on Silica Gel-60 F254plates (E. Merck) in sec-butanol/ethyl acetate/acetic acid/water(140/120/2/50). In Table I are the TLC results of the variousphallacidin derivatives. Because the ethylendiamine phallacidin has afree amino group, it is immobile on silica gel in the solvent systemsused. The disappearance of the mobile spot containing phallacidin methylester indirectly indicated the reaction with ethylenediamine wassuccessful. The major fluorescent spot of the NBD-Cl phallacidinreaction was eluted from silica gel and the 240-320 nm absorptionspectrum was measured. It showed both toxin and fluorophore in the samespot. The absorption spectrum of N-Ph, see step D, was from 240-320 nm.The peak at 290 is characteristic of the thioether tryptophan linkageand indicates that this portion, necessary for toxin activity, is stillintact. This spectrum is equivalent to that of phalloidin.

                  TABLE I                                                         ______________________________________                                        THIN LAYER CHROMATOGRAPHY                                                     Compound            R Values                                                  ______________________________________                                        Phalloidin          .32                                                       Phallacidin         .16                                                       Phallacidin Methyl Ester                                                                          .37                                                       Ethylenediamine Phallacidin                                                                       <.05                                                      NBD-Phallacidin     .42                                                       ______________________________________                                    

Phallotoxins stabilize and bind to F-actin. Therefore dialysis andviscosity experiments were performed to test whether the amino alteredphallacidin derivative still retained this property. If so, it could beexpected to show binding to actin upon dialysis and promote an increasein viscosity of actin solutions. The dialysis measurements did show N-Phbinding to actin with respect to ovalbumin. The viscosity measurementspresented in Table II indicated that N-Ph increased the state of actinpolymerization. The toxin was subsequently labeled with NBD and wastested to determine if this labeled derivative retained biologicalactivity. The impure NBD-Ph toxin fraction successfully stained actincables in both fixed and living tissue culture cells and in perfusedcells of the algae Chara australis. It would be competitively inhibitedfrom actin binding by an excess of unlabeled phalloidin.

The absorption and emission spectrum of the purified fluorescentpurified NBD-ethanolamine has extinction coefficients ε470=2.4×10⁴/1-M-cm and ε346=9×10³ /1-M-cm. Assuming that the fluorophore extinctioncoefficients remain the same after coupling to the toxin and are knownexactly, a ratio of 1.27 mol toxin/mol NBD was calculated. In otherwords, at least 79% of the toxin from step F is labeled. The cumulativereaction yield was 13% to N-Ph. Typical yields of NBD reaction variedbetween 20-25%.

In the same manner as phallacidin, any compound corresponding to theformula: ##STR4## wherein R is --OH, and R₁, R₂, and R₃ areindependently selected from the group consisting of --H and --OH, can bereacted to form a compound corresponding to the formula: ##STR5## whereR₁, R₂ and R₃ are independently selected from the group consisting of--H and --OH,

where Z is ##STR6## where x is 0 or a whole number, preferably 1-10,most preferably 2-10, ##STR7## or the moiety where W or Y are organicfluorophores such as resulting from reacting a terminal amino group orsulphydryl group with compounds such as fluorescein-5-maleimide or thelike. Other examples of W include the reaction products of the terminalamino group with compounds such as lissamine rhodamine B sulfonylchloride, dansyl chloride, fluorescamine, fluorescein-isothiocyanate, 5or 6 (3,5-dichlorotriazinyl)amino fluorescein (5 or 6 DCTAF), and likecompounds where the fluorescein associated with the reading group isreplaced by moieties derived from rhodamine B, tetramethyl rhodamine,eosin and the like. [2,3,4-ij:5,6,7,-i'j']diquinolizin]-3-one, 2',3',6',7'-12',13',16',17'octahydro-4(or 5)-isothiocyanato-spiro[isobenzofuran-1(3H)].

The amino group can be inserted into the acid phallatoxin molecule by,for example, reacting the carboxyl group in a manner to insert the aminogroup e.g. ##STR8## The terminal amino group can be converted to themercapto group or the mercapto group can be inserted in the molecular byreacting the acid group or its methyl ester with lysine or a similardifunctional compound. The individual reaction steps required to formthe compounds of the formula IV are all within the skill of the art. Theresultant compounds of Formula IV are useful in the same manner as theNBD-phallacidin specifically exemplified herein, and are useful to studyF-actin and G-actin oligomers in vivo and in vitro.

METHOD OF STAINING LIVING CELLS

Living cells were permeabilized by standard techniques as in the methodof M. R. Miller et al. as described in Experimental Cell Research, 820,421-5 (1979) using a solution of 14-40 μg/ml of palmitoyllysophosphatidyl choline (Sigma) in a high glucose buffer, buffer A,consisting of: 120 mM NaCl₂, 15 nM glucose, 10 mM Hepes, 4.4 mM NaHCO₃,mN KCl, and 1 mM Na₂ PO₄ adjusted to pH 1,7 with 1N NaOH. Two stainexposure methods were developed. In method I the cells were washed fourtimes with buffer A to remove the lysolecithin after two minutes andthen stained for five minutes with 5 ng of the purified NBD-phallacidinin 350 μl of buffer A at room temperature. The cells were then washedthree times and viewed within 1 hour. In method II cells were washed onetime and then stained for five minutes with NBD-phallacidin dissolved inDMEM plus 10% FCS in the 37° C. incubator. These cells were thentransferred to warm medium and held in the incubator.

For tests of viability flurescin diacetate (Sigma) in stock solution at2 mg/ml actone was applied at 0.5 μl per ml of cell medium and cellswere inspected promptly for reactive staining.

LABELING OF ACTIN CABLES IN ACTIVELY STREAMING CHARA AUSTRALIS

Internodal cells of Chara australis were isolated from the plant andwere placed in petri dishes containing artificial pond water. Tofacilitate microscopic viewing, "windows" through the dense chloroplastlayer were produced by the technique of E. Kamitsubo as described inExperimental Cell Research, 74, 613-6 (1972). Within 10 days the actincables returned to the window area, and at that point the cells wereperfused following the technique of M. Tazawa et al. as described inCell Structure Function, 1, 165-176 (1976), (13) with 1.5 μg of NBD -Phdissolved in 50 μl of Tazawa's perfusion fluid consisting of 30 mMhepes, 5 mM EGTA, 6 mM MgCl₂, 1 mM ATP, 23.5 mM methanesulfonic acid,and 250 mM sorbitol (pH-7 as adjusted with KOH). Photographs wererecorded approximately one hour after perfusion.

Fluorescence photomicrographs were taken using epifluorescentillumination from either a mercury lamp or the 448 nm line of an argonlaser. The last-illuminated photomicrographs often appear somewhatmottled due to optical interference.

Fixed fibroblastic cells of various common forms stained withNBD-phallacidin display the general features of the actin cytoskeletonwhich were expected from recent indirect immunofluorescence experimentswith actin antibody. To illustrate, with fixed and stained chick embryofibroblasts in NBD-phallacidin fluorescence, the expected actin stressfibers and fiber bundles are clearly marked. There is no nuclearstaining.

Competitive staining experiments on CEF designed to test specificitywere conducted under 1500 magnification. The cells in one group werestained by the standard procedure with NBD-phallacidin analog and in asecond group with an accompanying 50 fold excess of unlabeledphalloidin. 3T3 cells (not shown) give similar results. The CEF cellsdemonstrate diffuse membrane staining, and an abundance of stainedcables. The paired dish, stained in the presence of competing phalloidinshowed only very weak levels of possibly non-specific staining. Thespecificity of the fluorescent labeled toxin was thus established.

For comparison, 3T3 cells labeled with free NBD-Cl show prominentnuclear and very little peripheral fluorescent. NBD-ethanolamineproduces low levels of diffuse fluorescence. The features of thesestains are clearly distinguishable from the toxin staining.

Living fibroglastic cells permeabilized with lysolecithin and stainedwith NBD-phallacidin have fluorescence and phase contrast images thatare virtually indistinguishable from similar cells that have been fixedand stained. However, detailed microscopic examination (1500magnification) of the fluorescence of these cells does show that itdepends sharply on depth and plane of focus, indicating the threedimensional features of the actin cytoskeleton, whereas the fixed cellsused in the present invention tend to collapse somewhat. There is also atendency for some microfilament bundles to display a more feathery andslightly curvilinear appearance than is usually observed in fixed cells.

Prominent actin structures in the ruffles at the perimeter of an activelamellipodium were examined. Most of the actin filament bundlesextending into the lamellipodium from the cell center appear toterminate well before reaching the periphery.

An MEF cell, or cells, was photographed in mitosis. Here the cleavagefurrow associated actin separating the two cell volumes was clearlyvisible. No fibrous organization of the actin was visible in any planeof the fluorescence images.

Three NBD-phallacidin fluorescence images of MEF's (1250 magnification)illustrated the diversity of virtually unaltered cytoskeletal structuresthat can be captured. In one the actin "Cellular geodome" sometimes seenin rounded cells was preserved. In another a round cell that has justbegun to spread shows prominent actin structures in its regularlyruffled border and evidence of early axial alignment of central actincables. One could imagine that the cytoskeletal structure shown in thethird represented a later stage of development. A prominent cross bandedactin network terminates the central region of quasi-radial actinbundles.

Effects on living fibroblasts of the permeabilization treatment andtoxin staining were appraised for structural perturbations and reductionof cell viability over both short and long times by microscopicobservations and by fluorescein diacetate (FDA) reactive staining. Thiswell established staining method provided a convenient objective measureof the recovery of membrane integrity and cell viability and isdemonstrated by B. Rotman and B. W. Papermaster in Proc. Nat. Acad. Sci.USA, 55, 134-141 (1966).

M. R. Miller et al., supra, had established by assays of DNA synthesisand membrane leakage that their lysolecithin permeabilization procedure(without toxin staining) left cells viable for at least sixty minutesafter permeabilization. In the present invention it was found that insurveying a wide range of conditions that short term recovery, asdefined by FDA staining and cell appearance, was quite sensitive todetails of the permeabilization procedure and subsequent conditionsduring recovery. The viability was retainable at toxin levels necessaryto provide adequate actin staining. FDA staining after sixty minutes wasretained even with an indrease of the NBD-phallacidin dose to 2-3 μg/ml;where the cells were incubated with NBD-phallacidin in medium withserum.

In summary, the effects on living fibroblasts of the lysolecithinpermeabilization process and accompanying toxin staining of the presentinvention include the following: (1) The doses required for adequatestaining of virtually all cells in a dish introduces no initial visibleperturbation of the cell structure. (2) Viability of toxin stained cellsas assayed after 60 minutes by FDA fluorescence generally approximatelyor exceeded 33% but is variable and sensitive to the lysolecithinpermeabilization treatment. (3) Several fold increases of the dose oflysolecithin above the standard levels induces rapid cell loss. (4) In afew cases the MEF's concentrate the fluorescent marker into internalspherical structures that may be vesicles. Similar structures haverecently been observed subsequent to fluorescent actin microinjection byT. E. Kreis et al in Proc. Nat. Acad. Sci. USA, 76, 3814-3818 (1979).Although long term studies are incomplete and variable in their results,the observations from the present invention together with those of M. R.Miller et al., supra, suggest that some of the permeabilization andstaining procedures leave cells viable by the FDA assay for many hours.

The following discussion deals with working actin cables in living Charaduring cytoplasmic streaming. The giant cells of Characean algae showrapid rotational cytoplasmic streaming. Electron and fluorescencemicroscopic studies employing myosin fragments having identifiedsubcortical actin cables which are believed to participate in thegeneration of the streaming in these cells. These cables formunambiguous structures attached to the chloroplast files. Living,streaming Chara cells perfused with NBD-phallacidin show fluorescentsubcortical actin cables whereas control cells perfused with NBD-Cl andNBD-ethanolamine show no fluorescent cables. Structural features of theactin cable network marked by NBD in these living cells confirm featurespreviously observed in fixed cells marked with myosin fragments such asreported by R. E. Williamsom in Nature, 248, 80102 (1974).

The fluorescent stain on some cables appears somwhat fuzzy. Electronmicroscopy has previously revealed endoplasmic filaments in associationwith actin cables. Unresolved fluorescence in one instance may consistof aggregates of these submicroscopic filaments which have boundNBD-phallicidin, but artifacts have not been excluded.

It is to be emphasized that the photomicrographs made showed cells whichwere actively streaming at near normal rates. Labeling with myosinfragments stops streaming. The fluorescent marked cables during thecontinuation of cytoplasmic streaming were observed in the presentinvention for several hours following NBD-phallacidin perfusion. Thiscapability for observing the actin cytoskeleton in living cells has madepossible the studies of the dynamics of cytoplasmic streaming in futurestudies.

In conclusion, the present invention has shown in both tissue culturecells and algal cells that NBD-phallacidin labels a variety ofstructures known to consist of F-actin. Moreover, phalloidin competitionexperiments and control stainings show that the staining is specific forF-actin.

The present invention has observed with NBD-phalladicin large amounts ofdiffuse and fibrous actin that appear to be membrane associated. Becausethe diffuse staining is similar in living cells that are permeabilizedand stained, and in fixed and stained cells, it seems unlikely to be dueto an artifact. Of course, phallacidin does stabilize F-actin and maytherefore induce association of dissolved G-actin or F-actin fragmentswith membrane-associated F-actin. Similarly, cross-linking by thefixative in fixed and stained cells may accomplish a similaraggregation.

The cell nuclei are not stained by NBD-phallacidin in either live orfixed tissue culture cells. In contrast, photographs of actin antibodystaining show staining of cell nuclei as shown by W. W. Franke et al.,in J. Cell Biol., 81, 570-580 (1974). This difference implies thateither the nuclear actin is entirely monomeric G-actin which does notbind the toxin or that the actin antibodies have stained nucleinonspecifically.

Through the use of NBD-phallacidin florescent stain all of thestructures in live cells can be observed with as excellent resolution asthose in fixed cells such as stree fibers, ruffled borders, and diffusemembrane actin. Since the cells are not fixed, there is no distortionintroduced by the fixation procedure. One might worry that thelysolecithin treatment may alter cytoskeletal structure, but after thestaining treatment of the present invention no gross morphologicalalterations in structure of various known cell forms was observed incells in which the actin distribution was observed. Because the toxinenters the cells uniformly by diffusion through the permeabilizedmembrane, its concentration and rate of delivery are smoothly regulated.It is believed that in this way cellular damage and artifact formationare minimized in comparison with microinjection. Perhaps it is thisdifference of treatment that has prevented formation of the localizedaggregates of actin seen by Wehland et al. in Proc. Nat. Acad. Sic. USA,74, 5613-7 (1977), upon microinjecting phalloidin into live cellsfollowed by fixing and indirect actin staining.

Both phallacidin and lysolecithin permeabilization do perturb livingfibroblasts but effects on structure have been unobservable by lightmicroscopy and viability loss appears relatively slow. The effects ofNBD-phallacidin on the dynamics of cytoskeletal change, the effects onviability or the optimization of the staining procedures to minimizeperturbation of cellular processes have not fully been characterized.Nevertheless the fluorescent toxin of the present invention should proveuseful as a probe to observe some aspects of cytoskeletal change inanimal cells.

Observations of actin filament configurations and movements duringcytoplasmic streaming in the living, perfused algal cells of Charaaustralis were made possible by use of NBD-phallacidin. This experimentillustrates the usefulness of the toxin as an in vivo actin marker thatenables observation of previously inaccessible dynamic processes. Suchobservations must be interpreted carefully because phalloidin andphallacidin are known to stabilize F-actin and hence may alter thenative degree of actin polymerization in live cells. In the Charaexperiments the toxin is not disruptive and this observation suggeststhat depolymerization of actin is not an essential step in the pumpingof cytoplasmic streaming in these cells.

Fluorescence labeled phallacidin is a preferred actin stain, becausephallacidin is extremely stable and is relatively abundant in Amanitaphalloides its availability is assured. The low molecular weight of thelabeled toxin allows its introduction into slightly permeabilized livingtissue cells. Because permeabilization procedures are applicable enmasse to an entire culture dish population, the overall procedureprovides a large sampling of a cell population in contrast withmicroinjection techniques.

We claim:
 1. A compound corresponding to the formula: ##STR9## whereinR₁, R₂ and R₃ are independently --H or --OH and Q is selected from thegroup consisting of: ##STR10## where x is 0 or a whole number.
 2. Thecompound of claim 1 wherein x is 1 to
 10. 3. The compound of claim 2wherein x is 2 to
 10. 4. The compound of claims 1, 2 or 3 wherein Q is(a).